Cells communicate with and respond to their environment by receiving and processing extracellular signals. These signals take the form of growth factors, hormones, cytokines, and peptides which bind to and activate specific plasma membrane receptors. The activated receptors trigger intracellular signal transduction pathways which culminate in a wide range of cellular responses affecting gene expression, protein secretion, cell cycle progression, and cell differentiation. Initial events in signal transduction require the proximity of intracellular signaling proteins to the cytosolic domains of activated plasma membrane receptors. These intracellular membrane-associated signaling proteins couple the activated receptor to downstream second messenger systems and play a key role in the regulation and coordination of complex, multiprotein signal transduction pathways. Recently, a conserved protein domain called the PDZ domain has been identified in various membrane-associated signaling proteins. This domain has been implicated in receptor and ion channel clustering and in the targeting of multiprotein signaling complexes to specialized functional regions of the cytosolic face of the plasma membrane. (For review of PDZ domain-containing proteins, see Ponting, C. P. et al. (1997) Bioessays 19:469-479.)
PDZ domains were named for three proteins in which this domain was initially discovered. These proteins include PSD-95 (postsynaptic density 95), Dlg (Drosophila lethal(1)discs large-1), and ZO-1 (zonula occludens-1). These proteins play important roles in neuronal synaptic transmission, tumor suppression, and cell junction formation, respectively. Since the discovery of these proteins, over sixty additional PDZ-containing proteins have been identified in diverse prokaryotic and eukaryotic organisms. A large proportion of PDZ domains are found in the eukaryotic MAGUK (membrane-associated guanylate kinase) protein family, members of which bind to the intracellular domains of receptors and channels. However, PDZ domains are also found in diverse membrane-localized proteins such as protein tyrosine phosphatases, serine/threonine kinases, G-protein cofactors, and synapse-associated proteins such as syntrophins and neuronal nitric oxide synthase (nNOS). Generally, about one to three PDZ domains are found in a given protein, although up to nine PDZ domains have been identified in a single protein.
X-ray crystallography has shown that PDZ domains are generally compact globular structures containing about 80 to 100 amino acids which form six .beta.-strands and two .alpha.-helices. PDZ domains tend to be rich in glycine residues which introduce turns in the polypeptide chain and promote compaction and stability of the folded polypeptide. In particular, a glycine-aspartic acid (GD) dipeptide and an asparagine residue which occurs six residues thereafter are highly conserved in nearly all PDZ domains and are important for domain structure. PDZ domains bind to a tripeptide motif containing valine and serine or threonine. Most ligands which bind PDZ domains contain this motif, although some ligands lack this motif or contain conservative substitutions therein.
Members of the MAGUK protein family play an important role in the clustering of neurotransmitter receptors and channels, and this clustering is essential for neuron function (Ponting, supra). In the presence of a neurotransmitter, the PDZ domains of PSD-95, PSD-93, SAP-97 (synapse-associated protein 97), SAP-102, and chapsyn 110 bind to the cytosolic C-termini of N-methyl-D-aspartate (NMDA) neurotransmitter receptors and Shaker-type potassium channels, causing them to cluster. In addition, interaction of the PDZ domain of PSD-95 with that of nNOS anchors the latter in proximity to the NMDA receptors.
In the nematode Caenorhabditis elegans, the PDZ-containing protein, LIN-7, is required during development for the specification of cells which give rise to the vulva (Simske, J. S. et al. (1996) Cell 85:195-204). These precursor cells form an epithelium in the sexually immature nematode. A diffusible epidermal growth factor (EGF)-like signal is secreted in the vicinity of the epithelium. Cells in sufficiently close proximity to the signal respond by activating an EGF-like receptor tyrosine kinase/Ras-mediated signal transduction pathway. Responding cells subsequently give rise to generations of progeny cells which differentiate to form the vulva. The EGF-like receptor (EGFR) is localized to epithelial cell junctions, and this localization is essential for signal transduction and vulva formation. The LIN-7 gene is required for EGFR localization and encodes a 297 amino acid protein (SEQ ID NO:3) which contains a PDZ domain from amino acid 199 to amino acid 280.
PDZ-containing proteins are likely involved in disorders associated with defective cell signaling (Ponting, supra). As discussed above, PDZ domains have been shown to play important roles in development, and in fact, the gene encoding the PDZ-containing protein, LIM kinase 1, is deleted in patients with Williams syndrome, a complex developmental disorder. PDZ-containing proteins have also been implicated in oncogenesis. For example, mutations in Drosophila Dlg causes neoplastic transformation of epithelial cells, and N-terminally truncated forms of the PDZ-containing protein, Tiam 1, are highly tumorigenic in nude mice. In addition, mutations that block clustering of neuronal receptors and channels cause perinatal lethality in mice, suggesting that the clustering function of neuronal MAGUK proteins is critical.
The discovery of a new cell junction PDZ protein and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of cancer, neurological disorders, and developmental disorders.